PT2289540E - Alpha-1-antitrypsin for use in the treatment of chronic fatigue syndrome - Google Patents

Description

ΡΕ2289540 1

DESCRIPTION

" ALPHA-1-ANTITRYPSIN FOR USE IN TREATMENT OF CHRONIC FAIL SYNDROME " The present invention relates to the use of alpha-1-trypsin for the preparation of drugs effective for the treatment of chronic fatigue syndrome.

Background Chronic fatigue syndrome (CFS) is a complex disorder, defined by the international Fukuda criterion (Fukuda K, et al., "The chronic fatigue syndrome: a comprehensive approach to its definition and study", Ann Intern Med. 1994 ; 121: 953-959) under the supervision of the Atlanta Center for Disease Control (CDC). According to these criteria, the diagnosis of CFS is based on the satisfaction of two main criteria and the coexistence of a minimum of four minor criteria.

Main Criteria 1. Persistent physical and mental fatigue for at least six months, or intermittent, of new or defined first symptoms, which does not result from recent efforts, is not alleviated by rest, and becomes 2 ΡΕ2289540 worse with activity, and which causes a substantial reduction in the previous levels of daily activity of the patient, which ultimately the patient can not exceed. 2. Exclusion of other disorders that may potentially cause chronic fatigue, such as endocrine, infectious, neoplastic and / or psychiatric disorders.

Minor criteria

Four or more of the following minor criteria should be concurrently present, lasting for all six months or more after the onset of fatigue: - Concentration weakening or short-term memory. - Odynophagia - Cervical or axillary painful adenopathies - Myalgia - Polyarthralgia without signs of inflammation. - Headaches of early or recent symptoms with characteristics other than normal. - Not very refreshing sleep. 3 ΡΕ2289540 - Post-use disease lasting more than 24 hours.

Among the disorders that can be confused with CFS is fibromyalgia (FM), which is a syndrome characterized by symptoms of generalized chronic musculoskeletal pain, which is not of joint origin. According to the classification criteria of the American College of Rheumatology (The American College of Rheumatology, 1990, "Criteria for the Classification of Fibromyalgia: Report of the Multicenter Criteria Committee, Arthritis Rheum 1990; 33 (2): 160- 172) the two basic characteristics for the diagnosis of FM are: 1) the presence of generalized pain that lasts for three months; 2) abnormal digital pressure sensitivity of approximately 4 kg in at least 11 of 18 specific points, known as " tender points ". In addition to pain, patients with fibromyalgia experience some of the following symptoms: sleep disorders, irritable bowel syndrome, ankylosis and stiffness, head or face pain, abdominal disease, irritable bladder, paresthesia, numbness or itching, chest pain, and costochondralgia (pain in the muscle where the ribs attach to the sternum), dizziness and nausea, etc. Symptoms tend to fluctuate and do not necessarily occur simultaneously. 4 ΡΕ2289540 FM and CFS are two different disorders but with similar presentation and symptoms, often confusing non-experts, although they may coexist in many patients. Although 80% of CFS sufferers meet the FM classification criteria, although 7 to 10% of FM patients meet those for CFS. The differential diagnosis between the two and eliminating other possible causes of pain and fatigue is fundamental for a correct diagnosis and therapeutic approach. CFS predominantly affects young adults with a peak of early symptoms in their 20s and 40s. And 2-3 times more common in women than in men (Lloyd AR, et al., &Quot; Prevalence of chronic fatigue syndrome in an Australian population ", Med J Aug 1990; 153: 522-528), although this proportion may be due to women seeking medical care at all levels more frequently (Henderson AS., "Care-eliciting behavior in man, J Nerv Ment Dis 1974; 159: 172-181). The prevalence of CFS in the population is between 0.4 and 2.5% (White PD, et al., Protocol for the PACE trial: a randomized controlled trial of adpative pacing, cognitive behavior therapy, and graded exercise, supplements to standardized specialist medicai care versus standardized specialist medical care alone for patients with the chronic fatigue syndrome / myalgic encephalomyelitis or encephalopathy, BMC Neurol 2007, 7: 6). In the United States and the United Kingdom, four studies gave an estimate of 0.2% to 0.7%, in other words, 200 to 5 ΡΕ2289540 700 cases per 100,000 people. In Japan a prevalence of 1.5% was recorded. In general, estimates of the prevalence of CFS were between 0.5% and 2.5% in primary care settings, depending on the intensity of the medical, psychiatric, and laboratory evaluation (The Royal Australasian College of Physicians, " Chronic Fatigue Syndrome " Clinical Practice Guidelines 2002). The prognosis for recovery of CFS is extremely poor and there is currently no universal treatment that has been shown to be an effective option for treating CFS (Hill NF, et al., "Natural history of severe chronic fatigue syndrome", Arch Phys Med Rehabil 1999; 80 (9): 1090-1094). Therefore, in the current state of affairs, the primary therapeutic goal is based on symptom relief. Some of the treatments offered include: cognitive-behavioral therapy, graduate exercise therapy, pharmacological intervention (such as antiviral, antidepressant, sedative, analgesic, anti-inflammatory and other drugs) and nutritional supplements. However, these interventions do not often produce the minimal benefit considered necessary in many CFS patients (Am J Psychiatry, 2003; 160 (2): 221-236 / Rimes KA, et al. &Quot; Treatments for Chronic Fatigue Syndrome ", Occupational Medicine 2005: 5 (1); 32-39). It is therefore clear that there is a need for effective medicines for the treatment of CFS. 6 ΡΕ2289540 SFC is a multisystem disease of which the etiology or the triggering factor is unknown, although there are several hypotheses regarding the causative agents: genetic defects, central nervous system abnormalities, neuromuscular and metabolic irregularities, psychological factors, toxic agents, infections and immunological imbalances due to chronic activation of the immune system (Afari N, et al., " Chronic fatigue syndrome: a review ", Am J. Psychiatry, 2003; 160 (2): 221-236). Specifically, based on the chronically activated immune state, some authors have suggested that the clinical and immunological abnormalities observed in CFS may include defects in the interferon-induced 2-5A defensive pathway (Englebienne P, et al., &Quot; Chronic Fatigue Syndrome. ; CRC Press LLC, 2002).

Interferons (IFs) are proteins naturally produced by the immune system in response to external agents such as bacteria, viruses and parasites, and cancer cells. The two most significant products for IF stimulation are protein kinase R (PKR) and ribonuclease L (RNase L). PKR inhibits the translation of the viral mRNA while the RNase L turns off the dsRNA. The ultimate goal of both proteins is to induce apoptosis of infectious agents.

In 1994 Suhadolnik, et al. (S96-S104) found that peripheral blood mononuclear cells (PBMCs) were found to be the most effective inhibitors of peripheral blood mononuclear cells (PBMCs) of patients with CFS had an overactive RNase L with a 37 kDa molar mass, produced by proteolysis of the native 83 kDa RNase L form. Later, De Meirleir, et al. (" A37 kDA 2-5A binding protein as a potential biomarker for chronic fatigue syndrome ", Am J Med 2000; 108 (2): 99-105) has observed that the ratio of 37 kDa molecule concentration to 83 kDa in PBMC was useful for differentiating CFS patients from those suffering from FM or major depression.

Patients with CFS have many symptoms that are characteristic of dysfunctions in the transport of ionic channels. The potential for ion channel disruption in CFS patients was taken into account when it was determined that the RNase L (RLI) inhibitor belonged to the superfamily of ABC ion channel transporters. RLI deactivates RNase L by combining the ankyrin domains present in RNase L. The elimination of the ankyrin domain during fragmentation of RNase L, seen in CFS patients, has suggested that these ankyrin fragments may be able to interact and disrupt normal ionic channels. A dysfunction of these transporters would explain many of the symptoms found in CFS patients: night sweats, sarcoidosis, chemical hypersensitivity, macrophage dysfunction, impaired immune system, discontinued monoamine transport, increased pain sensitivity, Th2 dominance, central nervous system abnormalities , vision problems, loss of potassium in the muscles, ΡΕ2289540 transient hypoglycemia and depression (Englebienne P, et al., " Interactions between RNase L, ankirin domain and ABC transports as a possible origin of pain, CNS and immune disorders of chronic fatigue immune dysfunction syndrome, J Chronic Fatigue Syndrome 2001; 8 (3/4): 83-102). Elastase, cathepsin-G and m-calpain are enzymes capable of causing the proteolysis or fragmentation of Rnase L (Englebienne P, et al., &Quot; Interactions between RNase L, ankyrin domains and ABC transports as a possible origin of pain, ion transport, CNS and immune disorders of chronic fatigue immune dysfunction syndrome, J Chronic Fatigue Syndrome 2001, 8 (3/4): 83-102 / Demetre E, et al., " Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients ", J Biol Chem 2002: 20; 277 (38): 35746-35751). These three proteases are involved in defense mechanisms against pathogens and in inflammatory processes, and are therefore often found at abnormally high concentrations during an inflammatory response. In the case of CFS, it was found that patients suffering from such a disorder usually had high concentrations of elastase (Demetre E, et al., &Quot; Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients ", J Biol Chem 2002 : 20; 277 (38): 35746-35751 / Nijs J, et al. &Quot; Chronic fatigue syndrome: performance exercise related to immune dysfunction, Med. Sports Exercise 2005; 37 (10): 1647-1654).

Demetre E, et al. demonstrated that elastase has a significant role for RNase L degradation in 9 ΡΕ2289540 when they proved that a specific elastase inhibitor was able to inhibit to a large extent the proteolysis of RNase L in a PBMC culture of CFS patients.

Faced with the need to find effective drugs for the treatment of CFS, the inventors have carried out extensive, in-depth investigations and tests that have resulted in the present invention, which is based on the use of alpha-1-antitrypsin (AAT) for the preparation of drugs for the treatment of CFS.

Description of the Invention

Alpha-1-antitrypsin (AAT) is a glycoprotein secreted in hepatocytes, and is normally present at high concentrations in serum and most tissues, where it acts as a serine protease inhibitor. Reference values for AAT in the serum of healthy patients are 0.83-2.00 g / l (Kratz A, et al., Laboratory Reference Values, N Engl J Med 2004; 315 (15): 1548-1563). Apart from its activity as a protease inhibitor, AAT has been described as possibly having an important anti-inflammatory biological function, since it has a significant ability to inhibit many mediators of inflammation and oxidative radicals (Brantly M., " Alphal- antitrypsin: not just an antiprotease: extending the half-live of a natural anti-inflammatory molecule by conjugation with polyethylene glycol, Am J Respir Cell Mol Biol 2002; 27 (6): 652-654). 10 ΡΕ2289540 AAT deficiency is an inherited disease that primarily causes pulmonary emphysema in the early stages of adulthood (30-40 years). The second most frequent manifestation is liver disease, which can affect newborns, children and adults. Less frequent is an inflammatory skin disease known as neuromyzing panniculitis (American Thoracic Society / European Respiratory Society Statement: " Standards for the diagnosis and management of individuals with alpha-1-antitrypsin deficiency ", Am J Respir Crit Care Med 2003 ; 168: 818-900).

At present, there are therapeutic AAT concentrates prepared by the fractionation of human blood plasma which is used in AAT replacement therapy for the treatment of individuals with a deficiency of this protein and associated pulmonary emphysema. These concentrates were shown to be biochemically effective in raising serum AAT concentration above the minimum levels considered protective (11 μιηοΐ / ΐ) (Wewers MD, et al., &Quot; Replacement therapy for alpha-antitrypsin deficiency associated with emphysema ", New Engl J Med 1987; 316: 1055-1062). In addition, several clinical studies suggest that AAT replacement therapy is clinically effective in slowing the progression of pulmonary emphysema and decreasing mortality (Seersholm N, et al., &Quot; Does alpha-1-antitrypsin augmentation therapy slow the annual decline in FEV1 in patients with severe hereditary alphl-antitrypsin deficiency? &Quot; Eur Respir J 1997; 10: 2260-2263 / " The Alpha-1-Antitrypsin Deficiency 11 ΡΕ2289540

Registry Study Group, Survival and FEV1 decline in individuals with severe deficiency of alpha-1-antitrypsin ", Am J Respir Crit Care Med 1998; 158: 49-59).

Extensive clinical experience in AAT replacement therapy confirms that AAT therapeutic concentrates from human plasma have an excellent safety profile (Wencker M, et al., &Quot; Long term treatment of alpha-1-antitrypsin deficiency-related pulmonary emphysema with human alpha-1-antitrypsin ", Eur Respir J 1998; 11: 428-433 / American Thoracic Society / European Respiratory Society Statement: " Standards for the diagnosis and management of individuals with alpha -1-anti-trypsin deficiency ", Am J Respir Crit Care Med 2003; 168: 818-900).

To verify whether inhibition of elastase by AAT can prevent degradation of Rnase L, the inventors have carried out various studies using in vitro PBMC culture of patients with CFS together with AAT concentrates. Based on these studies, the inventors have established that the PBMC extracts from CFS patients showed high elastase activity, much more than the PBMC extracts from healthy subjects. PBMC extracts from six healthy subjects showed a mean elastase activity of 81U / mg extract (CV = 38.9), with a min-max of 51-125 U / mg extract, while the PBMC extracts of eight patients with CFS had a mean elastase activity of 322 U / mg extract (CV = 30.5), with a min-max of 193-453 U / mg extract. 12 ΡΕ2289540

The inventors also found that AAT was capable of substantially inhibiting the elastase intracellular activity of PBMC cultures from CFS patients. PBMCs from 10 CFS patients were cultured for 12 hours in the absence and presence of two different concentrations of AAT: 3 g / l and 6 g / l. The results obtained for the percent inhibition of elastase activity relative to the control without AAT were as follows: for the 3 mg / ml AAT culture, elastase intracellular activity was inhibited on average of -87.2% (CV = 0.09), with a minimum of -75.3 to -95.4%; for the 6 mg / ml AAT culture, the intracellular activity was inhibited on average of -91.0% (CV = 0.08), with a minima of -76.1 to -97.4%.

In addition, the inventors have established that AAT prevented 83 kDa RNase L degradation to generate the hyperactivative form of 37 kDa RNase L in PBMC cultures of CFS patients. PBMCs from two healthy subjects and the PBMCs from two CFS patients were cultured for 12 hours in the absence and presence of 3 g / l AAT. In the PBMC cultures of the two healthy subjects, no significant differences were observed in the analysis of RNase L degradation between the two cultures. Following the culture of PBMC without AAT, the RNase L ratio of 37 kDa to 83 kDa Rnase L was 0.3 and 0.4 while following the AAT culture, the RNase L ratio of 37 kDa to RNase L of 83 kDa was 0.2 and 0.3, respectively. Both values were below the 0.5 lookout threshold as 13 ΡΕ2289540 marking 83 kDa RNase L proteolysis. In the PBMC cultures of CFS patients, it was found that in the presence of AAT, degradation of RNase L decreased by 80%. Following non-AAT PBMC culture of the two CFS patients, the ratio of RNase L from 37 kDa to 83 kDa RNase L was 1.4 and 2.4 while following the culture with AAT, RNase L ratio of 37 kDa for RNse L of 83 kDa was 0.2 and 0.6 respectively.

The inventors have found that AAT has activated the expression of genes involved in the 2-5A synthetase pathway so that the administration of exogenous AAT can restore normal RNase L activity and prevent its proteolysis in the PBMCs of CFS patients. PBMCs from six CFS patients were cultured in the absence and presence of two different concentrations of AAT: 0.5 g / l and 3.0 g / l. In

then the RNA was extracted and analyzed with the " Genechips Human Genome U133 Plus 2.0 (Affymetrix ") system. The gene coding expression for the 2,5-oligoadenylate synthetase, the enzyme responsible for the synthesis of the 2-5A molecules, and thus the RNase L activators, increased 2.9 and 3.2 fold in cultures with 0.5 g / 1 and 3.0 g / 1 AAT, respectively, compared to cultures in the absence of AAT. Therefore, stimulation of 2,5-oligoadenylate synthetase expression by AAT can restore the activity of RNase itself and at the same time help prevent its proteolysis.

The inventors also found that AAT inhibited 14 ΡΕ 2289540 expression of metallothionines, and therefore the administration of exogenous AAT may reduce the activation of the proinflammatory pathways of the PBMCs of CFS patients. PBMCs from six CFS patients were cultured in the absence and presence of two different concentrations of AAT: 0.5 g / l and 3.0 g / l. Next, the RNA was extracted and analyzed with the Genechips Human Genome U133 Plus 2.0 (Affymetrix) system. The results were presented as the ratio between the expression of genes in the presence of AAT compared to the expression of genes in the absence of AAT. Expression of various genes encoding the metallothionines was reduced following cultures in the presence of AAT. Specifically, the expression of metallothionine 2A, metallothionine IX, metallothionine 1H, metallothionine 1F and metallothionine 1E decreased on average 2.3 and 4.5 fold in cultures with 0.5 g / 1 and 3.0 g / 1 of AAT respectively compared with cultures in the absence of AAT. As described, a deficiency in methionine reduces the production of proinflammatory cytokines (IL1b, IL6, TNFÎ ±) (Itoh N, et al., &Quot; Cytokine-induced metallothionine expression and modulation of cytokine expression by metallothionine ", Yakugaku Zasshi 2007 ; 127 (4): 65-694). Thus, inhibition of expression caused by AAT can produce the same effect and reduce proinflammatory pathways in PBMCs. In addition, metallothionins also play a very important role in the production of nitric oxide (NO), an immune mediator present in high concentrations in CFS patients (Kurup, RK et al., &Quot; Hypothalamic digoxin, cerebral Chemical dominance and myalgic encephalomyelitis "; Int J Neurosci 2003; 113 (5): 15, 228, 9540, 683-701). A decrease in NO production due to the reduction in AAT-induced expression of metallothiones may be another beneficial action of this protein in the context of CFS patients.

The inventors have also found that AAT has activated the expression of the gene encoding AAT so that the administration of exogenous AAT can promote its own expression in the PBMCs of CFS patients. PBMCs from six CFS patients were cultured in the absence and presence of two different concentrations of AAT: 0.5 g / l and 3.0 g / l. The RNA was then extracted and analyzed with Genechips Human Genome U133 Plus 2.0 (Affymetrix) system. The results were presented as the ratio between the expression of genes in the presence of AAT compared to the expression of genes in the absence of AAT. Expression of the gene encoding AAT (SERPINE 1) increased from 1.4 to 2.0 times on average on cultures with 0.5 g / 1 and 3.0 g / 1 AAT respectively, compared to cultures in the absence of AAT. Although AAT is synthesized primarily in hepatocytes, Perlmutter DH, et al. also described their expression in monocytes (" Cellular defect in alpha 1-proteinase inhibitor (alpha 1-P1) deficiency is expressed in human monocytes and in Xenopus oocytes injected with human liver mRNA ", Proc Natl Acad Sci USA, 1985; 82 (20): 6918-6921). Therefore, AAT expressed intracellularly and induced by the presence of AAT may have an inhibitory effect on the elastase of the PBMC cultures of CFS patients. Furthermore, it can not be deduced that AAT can directly 16 ΡΕ2289540 inhibit intracellular elastase upon entering PBMC cells, as described for other cell types (Zhang B, et al., &Quot; Alpha 1-antitrypsin protects beta-cells from apoptosis, Diabetes 2007; 56 (5): 1316-1323). Therefore, the two mechanisms of inhibition of elastase in PBMC cultures of CFS patients can coexist and even produce a cumulative effect.

These findings are even more surprising since the novel potential therapeutic applications of AAT-containing drugs originating from these experiments can in no way be related to the applications of this protein known hitherto which are strictly based on the compensation of the natural deficiency that presents as diseases (emphysema) or inflammatory skin disorders (panniculitis).

Although the inventors do not wish to feel bound to any hypothesis as to the form manifested by novel AAT-containing drugs in the treatment of CFS, they have established, without limitation, the hypothesis that AAT plays an important role in the control of the immune cells responsible for symptoms associated with CFS, and in regulating the expression of genes related to the immune system.

SFC can be treated with therapeutic AAT concentrates, purified from human plasma or produced by recombinant or transgenic technology. The 17 ΡΕ 2289540 treatment is also possible with plasma or other therapeutic products containing a sufficient amount of AAT to obtain a minimal dose.

As with other proteins, the presence of the complete AAT molecule is not thought to be necessary to achieve the desired result. Thus, molecules containing a partial sequence of amino acids derived from the corresponding sequence of the AAT molecule may be useful in the treatment of CFS. These molecules can be obtained from human plasma or produced by synthetic methods or by recombinant or transgenic technology. The present invention also relates to a method for the treatment of CFS which comprises administering a therapeutically effective amount of AAT in combination with one or more pharmaceutically inert or active carriers to a patient suffering from or at risk for developing CFS. The treatment regimen according to the invention includes periodic and repeated administration of AAT for the purpose of reducing or eliminating the symptoms of CFS. A dose of 6 mg or more AAT per kilogram (kg) of body weight infused at a frequency of between 1 and 31 days is considered sufficient for the treatment of CFS. A preferred dose of AAT would be between 15 and 360 mg per kg of body weight infused at a frequency of between 1 and 31 days. Yet a more preferred dose would be between 25 and 60 mg per kg 18æ2289540 of body weight every week or multiples of these amounts proportionally adjusted depending on the time interval expected until the next dose.

Alternatively, the present invention includes a treatment regimen established to achieve a desired serum AAT level up to eight times higher than base levels 24 hours after administration. AAT can be administered by parenteral injection and according to a preferred embodiment, administration takes place intravenously, although it may also be administered intramuscularly or intra-dermal. Alternatively, AAT can be administered by inhalation. Depending on the route of administration, the AAT preparation is made as a solution or suspension in a pharmaceutically acceptable carrier or carrier. Suitable examples of such vehicles include: water for injection, sterile water for injection and other aqueous vehicles (e.g., injectable sodium chloride, injectable Ringer's solution, injectable dextrose, injectable dextrose and sodium chloride, Ringer's lactate injectable); vehicles which may be mixed with water (for example ethyl alcohol, polyethylene alcohol, propylene glycol); non-aqueous vehicles (for example, corn oil, cottonseed oil, peanut oil and sesame oil). The need for and selection of other excipients, preservatives, buffer solutions, biocides and the like are within the scope of those skilled in the art and will depend upon a number of factors, including the system and route of administration, the shelf life required, and the conditions of transport and storage. EXAMPLE:

While awaiting the results obtained in vitro, the inventors, having obtained an authorization for compassionate use, administered an AAT-based preparation to a patient diagnosed with CFS.

A female patient was diagnosed with CFS in 2003 and met Fukuda's diagnostic criteria, eliminating other medical processes inducing chronic fatigue, such as endocrine, infectious, neoplastic and / or psychiatric disorders. Prior to initiating treatment with AAT concentrate, the patient had an elastase concentration in PBMC of 1459 U / mg (activity units per milligram of PBMC extract); in the functional reserve evaluation test, the patient had a maximum oxygen consumption of 17.2 ml / kh / min (63.5% of theory), a maximum power of 64 watts (54.0% of theoretical), a maximum heart rate of 149 (87.6% of theoretical): and in the study of neurocognitive dysfunction, showed very serious cognitive impairment. The patient was subjected to intravenous infusion therapy of AAT-based preparation (60 mg / kg body weight weekly) over a 20 ΡΕ 2289540 eight-week period. At the end of treatment, the patient had an elastase concentration in PBMC of 134 U / mg (activity units per milligram of PBMC extract); in the functional reserve evaluation test, the patient had a maximum oxygen consumption of 16.4 ml / kg / min (60.6% of theory), a maximum power of 85 watts (71.7% of theory), a maximum heart rate of 151 (88.8% of theoretical): and in the study of neurocognitive dysfunction, showed serious cognitive impairment. As a general conclusion, after treatment with the AAT-based preparation, the patient showed clear clinical improvement, returned to work, felt less fatigue and presented improved tolerance to physical exercise and slightly reduced cognitive dysfunction.

It has therefore been demonstrated that by the present invention, CFS patients can be effectively treated with drugs prepared on the basis of AAT. These patients would be affected by chronic inflammation of immune cells and, according to the present invention, AAT inhibits elastase thus avoids the degradation of RNase L, thereby preventing ion channel deregulation, which is supposedly responsible for the symptomatology associated with CFS . In addition, and according to results obtained in vitro, AAT can regulate the expression of particular genes associated with the immune system to restore normal functioning of the immune system and reduce the activation of the proinflammatory pathways. 21 ΡΕ2289540

Although the invention has been described relative to the examples of the preferred embodiments these should not be considered as limiting the invention which is defined by the broader interpretation of the following claims.

Lisbon, November 19, 2012

Claims (4)

A composition comprising alpha-1-antitrypsin or therapeutical forms thereof for use in the treatment of chronic fatigue syndrome, which may be administered to humans. A composition comprising alpha-1-antitrypsin for use according to claim 1, wherein said composition is administered in a treatment regimen of 6 mg or more of alpha-antitrypsin per kg of body weight at a frequency of between 1 and 31 days. A composition comprising alpha-1-antitrypsin for use according to claim 1, wherein the alpha-1-antitrypsin is purified from human plasma. A composition comprising alpha-1-antitrypsin for use according to claim 1, wherein alpha-1-antitrypsin is produced by synthetic, transgenic or recombinant technology. Lisbon, 19 November 2012 1 ΡΕ2289540 REFERENCES ISSUED IN THIS DESCRIPTION This list of references cited by the applicant is for the reader's convenience only. It is not part of the European patent document. While due care has been taken in compiling references, errors or omissions may not be excluded and the IEP declines any liability in this regard. Non-Patent Literature quoted in the Description * FiKKDA K & K * If you have more information on the patents, arta stesV. Mn Mem 1994, vol. 121, 953-35S. Drsteia te tae ClassiScsítars a! FIssofnytogis. Reporl of the Microsurgery of the Brain, Artattos RteiffJT 1 $ 8, 1990, vst 33 (2, 180-172 * LLO ¥ D AR et si. Preraitesee of chronic toftse syn ~ diwse ta sfs Aastraiíasi poptasSisrs. Meti J, Mtgusá 13SS, vol. 153, 522-358 - HENDERSOK AS, Care-eiteStag befcavfor in mm. J. Nerver, Vol. 15177-1S1 * WHHE PB et al, BMC NetsM 2® 7, and 7.8 * Chlorac Fatigsje Syratas. CIMsst Prsctice SiMs- rnm, * HILL RF st sL fcteaS híftoy o-í severe e-tetate &amp; -system sysdKsrse. Aidti-Pty &amp; Meti RehsM, 1998, (1998), 1SSS-1S94). AmJf &amp; 2Μ &gt;% νβΙ1 &quot; 2% 221-239 &quot; RSMESKA &quot; TABLE 1 - Results of the Sysi-sfeome. Orasjpa & Kotf, 2035, v. T. 5), 32-39 * FaSgue SyncSrome, PASSENGER PATIENT A BfotagSSaS Appísadt CRC Ptaas LL, 2882 * 8MHADOLMÊK «t st Upraguiaíto R © ftee 2-5A systostssRRsse L anSvirai pstowsy sssocásteí vsstt ctrctíss itaSgue syrtorame. Cysteine &lt; 1884, vot 18 888-8184 * BE «EIRLEiR st sL A 37 kDa 2-SA bmdtag ptesto s $ a potonito btodhettoeaf raraSwsrte steoíiSffiMigss syraftome. Am J Msd, 20, wd. 108 (2), 88-185: * RATZ: A et st LaPsnstesy Re ^ sretca Vabim1 N Eag JMed 2004, «A. 31S <1S |, 1S48-1S6S * SRANTLY Μ. Aiphysi-Syphilussei: netsuS sii: $ 3ip; cough: extssidltsg toe hatt-lto df 8 nsámê av &amp; itâasm-matexy mteoâ »&amp; $? ζζφ &amp; αη wfô »gycot; Am J RespO · CW M &amp; 8k $ 2082, vet 27 (8), 652-354), Amt &amp; at) TtKsradc Sade 'Surop ^ t Rampadray Soady dto-toe arago mannosis' of tadh4d (a) ptsa-1: SR8typs, csDgHKy, Am J Aq. 2333, v. 168, 818-956 * WEWER6 SiD st ai, R ás s nt nt nt nt T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 SR. Affect Big J Mean, 17, d. 316, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: a &quot; S &quot; &quot; Y &quot;. Sarcophagi, 1897. , pp. 18, 22, 288, 283, 248, 288, 288, 288, 288, 288, 288, and 308. AmJ Rssp k cm CsRsMsd, 19S8, woi, 158, 49-53 ♦ WSKCKB1! B5 et at, Long torra testment "fs4-phs-l-sr &amp; spsta {t ^ dera -physsma wita tiraast síp8s-1 -arstír / pssa. Ettr Raspir 4 1.88, s. 11.428 -433 (1H, s, N: st &amp; C &lt; 4 &gt; &lt; / RTI &gt; &lt; / RTI &gt; Yakuga &amp; Szmsfsi, 21367, voL 127 (4), 85-594 IRFTJP RR et al., Dichlorohydrazide, ssr-spherical stearic acid and myositis. M &amp; mcf, 20, vol. 113 (S), 683-701). TitooeMsfdefectfo topisa 1-pr ^ dnaseii: 1-PS (1-PS) dtokapter of Human Molecular Cytosis to Xenopyc ees ^ 1885, voi, 82 (.20), 89tS-S321 • ZMA14G B stito, Aiptisl ssilitilrypsta: prototo iso-to-tis Scra spoptoate. Diabetes, 2007, vol. SS (S), 1315-1323. E.R & LEB. RR: EP Interactions Betase, RRsse L, ssfcyrta doma® arai ABC transfection with passthrough cystitis, The present invention relates to a method for the preparation of the compounds of the invention. J Chrm, &lt; / RTI &gt; &lt; / RTI &gt;, 2301, vot 8 (3 (4), 83-102). (38), 35745-35751). In the present case, the present invention relates to a process for the preparation of a compound of the formula (I) 20C35, VSL 37 (43), 1547-1554